ABSTRACT MEYERS, EMILY ANN. Evaluation of Fungicide Sensitivity of United States Blumeria Graminis F. Sp. Tritici Population
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ABSTRACT MEYERS, EMILY ANN. Evaluation of Fungicide Sensitivity of United States Blumeria graminis f. sp. tritici Population. (Under the direction of Dr. Christina Cowger). Wheat powdery mildew, caused by Blumeria graminis f. sp. tritici, is managed primarily with wheat (Triticum aestivum) cultivar resistance and foliar fungicides in the United States (U.S.). Both management strategies are prone to deterioration and defeat simply due to the biology and disease cycle of this pathogen. In the U.S., host resistance gene effectiveness has been measured once a decade for the past 30 years. Despite high levels of fungicide insensitivity in other global cereal powdery mildew populations, fungicide sensitivity in the U.S. B. graminis f. sp. tritici population has never been evaluated. Formulated fungicide products from three mode-of-action categories are labelled for use on wheat in the U.S.: demethylation inhibitors (DMIs), quinone outside inhibitors (QoIs), and succinate dehydrogenase inhibitors (SDHIs). The study presented here sought to measure the sensitivity of the U.S. B. graminis f. sp. tritici population to fungicides from each of the three categories and determine the genetic causation of any observed decreases in sensitivity. Nearly 400 B. graminis f. sp. tritici isolates were collected from 27 fields in 15 states in the central and eastern U.S. in the 2013 and 2014 wheat growing seasons. Six fungicides were evaluated: 3 DMIs, (tebuconazole, prothioconazole, and metconazole), 2 QoIs (pyraclostrobin and picoxystrobin), and 1 SDHI (fluxapyroxad). Isolates were screened for fungicide sensitivity using a detached-leaf assay in which susceptible seedlings were sprayed with a concentrations of a formulated fungicide active ingredient, then cut into leaf segments which were floated atop water agar, and inoculated with a single B. graminis f. sp. tritici isolate. Leaf segments were later rated for fungal growth, then ratings were converted to EC50 values or estimates of the effective fungicide concentration at which an isolate’s growth was inhibited by 50%. Regional mean EC50s were most significantly different among the DMIs, with isolates originating from the eastern U.S. (especially the Mid-Atlantic and Great Lakes regions) exhibiting decreased sensitivity to DMIs compared to those from the central U.S. (Plains region, Arkansas, and Missouri). U.S. B. graminis f. sp. tritici isolates exhibited a diverse range in QoI sensitivity; however, differences in regional sub-populations were not as evident. Interestingly, on average isolates from the central U.S. were more SDHI insensitive than those from the Great Lakes and Southeast regions. Genetic sequencing of DMI and QoI fungicide target genes was completed to determine if any alterations correlated with observed differences in isolate sensitivity. In QoI target gene cytb, only synonymous mutations were found at previously identified mutation sites, further underlining the lack of major QoI insensitivity in the U.S. B. graminis f. sp. tritici population. In DMI target gene CYP51, 3 genotypes were found in the U.S. collection based on differences at the 136 locus: wildtype, Y136; mutant, F136; and heteroallelic (Het), Y136 & F136. In the DMI insensitive United Kingdom population, 2 CYP51 genotypes were identified involving changes at both the 136 and 509 loci: mutant, F136; Het-Het; Y136 & F136 + S509 & T509. Estimation of CYP51 gene copy number revealed 2-4 copies in isolates from both countries; however, CYP51 expression was twice as high in United Kingdom isolates as in U.S. isolates. Further research is necessary to fully understand the similarities and differences between these two populations. © Copyright 2020 by Emily Ann Meyers All Rights Reserved Evaluation of Fungicide Sensitivity of United States Blumeria graminis f. sp. tritici Population by Emily Ann Meyers A dissertation submitted to the Graduate Faculty of North Carolina State University in partial fulfillment of the requirements for the degree of Doctor of Philosophy Plant Pathology Raleigh, North Carolina 2020 APPROVED BY: _______________________________ _______________________________ Christina Cowger Barbara Shew Committee Chair _______________________________ _______________________________ Ignazio Carbone J. Paul Murphy ii DEDICATION To those that have supported me, especially when I needed it the most. iii BIOGRAPHY Emily was first exposed to agriculture while growing up on a family-run dairy farm in rural Wisconsin. After spending her childhood and teenage years feeding calves and picking stones, Emily attended Ripon College, a small liberal arts school in central Wisconsin, where she received a degree in Biology in 2013. Under the advisement of Dr. Mark Kainz, Emily attended two plant science summer research undergraduate internships where she was exposed to the field of plant pathology. She then joined the NC State plant pathology program in Fall 2013 and, after lab rotations, began her Ph.D. research on fungicide sensitivity of the U.S. wheat powdery mildew population under the direction of Dr. Christina Cowger. iv TABLE OF CONTENTS LIST OF TABLES ................................................................................................................... vi LIST OF FIGURES ............................................................................................................... viii Chapter 1: Literature Review ................................................................................................. 1 1.1 Global importance of wheat ........................................................................................... 1 1.2 Impact of Blumeria graminis on cereal crop production ................................................. 2 1.3 Cereal powdery mildew management and management challenges ................................ 3 1.3.1 Cultural management ............................................................................................ 4 1.3.2 Host resistance management ................................................................................. 5 1.3.3 Chemical management .......................................................................................... 6 1.3.3.1 Demethylation inhibitor fungicides............................................................... 7 1.3.3.2 Quinone outside inhibitor fungicides ............................................................ 8 1.3.3.3 Succinate dehydrogenase inhibitor fungicides .............................................. 8 1.4 Objectives of this study.................................................................................................. 9 References ......................................................................................................................... 11 Chapter 2: Sensitivity of the U.S. Blumeria graminis f. sp. tritici population to demethylation inhibitor fungicides ....................................................................................... 19 Abstract ............................................................................................................................. 20 2.1 Introduction ................................................................................................................. 21 2.2 Materials and Methods ................................................................................................ 25 2.2.1 B. graminis f. sp. tritici isolate collection ............................................................ 25 2.2.2 Detached-leaf fungicide sensitivity assay ............................................................ 26 2.2.3 Measuring sporulation as a component of fitness................................................. 27 2.2.4 Effective concentration calculations .................................................................... 28 2.2.5 Geographical comparisons .................................................................................. 29 2.3 Results ......................................................................................................................... 30 2.3.1 Range in EC50 value compared to European controls ........................................... 30 2.3.2 Geographic differences in DMI sensitivity .......................................................... 31 2.3.3 Sporulation variability and relationship to DMI sensitivity .................................. 33 2.3.4 Cross-resistance to DMIs .................................................................................... 34 2.4 Discussion ................................................................................................................... 34 2.5 Acknowledgements ..................................................................................................... 42 References ......................................................................................................................... 43 Chapter 3: United States wheat powdery mildew population sensitive to QoI and SDHI fungicides ............................................................................................................................... 59 Abstract ............................................................................................................................. 60 3.1 Introduction ................................................................................................................. 61 3.2 Material and Methods .................................................................................................. 66 3.2.1 B. graminis f.